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. 1999 Feb 16;96(4):1627-32.
doi: 10.1073/pnas.96.4.1627.

Human single-chain Fv immunoconjugates targeted to a melanoma-associated chondroitin sulfate proteoglycan mediate specific lysis of human melanoma cells by natural killer cells and complement

B Wang  1 , Y B Chen, O Ayalon, J Bender, A Garen
Affiliations

Human single-chain Fv immunoconjugates targeted to a melanoma-associated chondroitin sulfate proteoglycan mediate specific lysis of human melanoma cells by natural killer cells and complement

B Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Two antimelanoma immunoconjugates containing a human single-chain Fv (scFv) targeting domain conjugated to the Fc effector domain of human IgG1 were synthesized as secreted two-chain molecules in Chinese hamster ovary and Drosophila S2 cells, and purified by affinity chromatography on protein A. The scFv targeting domains originally were isolated as melanoma-specific clones from a scFv fusion-phage library, derived from the antibody repertoire of a vaccinated melanoma patient. The purified immunoconjugates showed similar binding specificity as did the fusion-phage clones. Binding occurred to human melanoma cells but not to human melanocytes or to several other types of normal cells and tumor cells. A 250-kDa melanoma protein was immunoprecipitated by the immunoconjugates and analyzed by mass spectrometry, using two independent procedures. A screen of protein sequence databases showed an exact match of several peptide masses between the immunoprecipitated protein and the core protein of a chondroitin sulfate proteoglycan, which is expressed on the surface of most human melanoma cells. The Fc effector domain of the immunoconjugates binds natural killer (NK) cells and also the C1q protein that initiates the complement cascade; both NK cells and complement can activate powerful cytolytic responses against the targeted tumor cells. An in vitro cytolysis assay was used to test for an immunoconjugate-dependent specific cytolytic response against cultured human melanoma cells by NK cells and complement. The melanoma cells, but not the human fibroblast cells used as the control, were efficiently lysed by both NK cells and complement in the presence of the immunoconjugates. The in vitro results suggest that the immunoconjugates also could activate a specific cytolytic immune response against melanoma tumors in vivo.

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Figures

Figure 1
Figure 1
Organization of the scFv targeting domain and Fc effector domain of an immunoconjugate molecule. VH and VL, heavy chain and light chain variable regions, respectively, derived from a scFv fusion-phage clone (7); a (G4S)3 polypeptide (curved line) links the VH to the VL region. H, hinge region of a human IgG1 Ig containing two disulfide bridges. CH2 and CH3, second and third constant regions of a human IgG1 Ig.
Figure 2
Figure 2
Anlysis of the immunoconjugate G71–1 by PAGE under reducing (lanes 1 and 2) and nonreducing (lanes 3 and 4) conditions. The immunoconjugate was espressed in CHO cells for lanes 1 and 3, and in Drosophila S2 cells for lanes 2 and 4.
Figure 3
Figure 3
FACS assays for binding of the immunoconjugate G71–1 to human melanoma cells and human control cells. (A) Melanoma lines, TF2. (B) Melanoma line A-2058. (C) Melanocytes. (D) Microvascular endothelial cells. (E) Fibroblast cells. (F) Transformed kidney line 293-EBNA. The cells were collected from a culture flask after detachment in a nonenzymatic dissociation medium (Sigma). The detached cells were either exposed to the immunoconjugate (outlined curve) or were not exposed (shaded curve). An increase in fluorescence after exposure to the immunoconjugate indicates that the cells bind the immunoconjugate. The number of gated events analyzed for each sample was at least 5,000. The immunoconjugate E26–1 also was analyzed by FACS and showed similar results as G71–1.
Figure 4
Figure 4
Immunoprecipitation of a human melanoma protein by immunoconjugate G71–1. The human melanoma line A2058 was equilibrated with the immunoconjugate and lysed with detergent, and the immunoconjugate-protein complex was isolated on protein-G beads and analyzed by PAGE using a 4–15% gradient gel. Lane M, Protein molecular weight markers. Lane 1, immunoconjugate alone. Lane 2, immunoconjugate with associated melanoma protein. The same experiment was done with immunoconjugate E26–1, which also immunoprecipitated a single protein with a molecular mass of 250 kDa.
Figure 5
Figure 5
Immunoconjugate-dependent lysis of melanoma cells by NK cells. The melanoma cell line A-2058 and the fibroblast cell control were labeled with the fluorescent dye Calcein-AM. The fraction of melanoma or fibroblast cells remaining intact after exposure to NK cells alone (A bars), or to NK cells with the immunoconjugate E26–1 (B bars), was measured by residual fluorescence. The ratio of NK effector cells to target cells (E/T) was varied from 3 to 20. Three complete sets of experiments were done for both the melanoma and fibroblast cells; for each experiment, the cytolysis assays were done in quadruplicate. The bars represent the average of the cytolysis assays for the three experiments, which generally agreed within 10%. The % cytolysis was calculated as described in Materials and Methods. Similar results were obtained with the immunoconjugate G71–1.
Figure 6
Figure 6
Immunoconjugate-dependent lysis of melanoma cells by complement. The procedure was as described in Fig. 5, except that human serum or purified rabbit complement components were used instead of NK cells. Immunoconjugate and complement reagents for each assay were as shown in Table 1.
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